# Hyaluronic Acid-Palmitate Nanoparticle Delivery of Carbonic Anhydrase Inhibitors Impairs Growth and Early Metabolism in Escherichia coli Through β- and γ-Carbonic Anhydrase-Associated Processes

## TL;DR

This paper shows that using hyaluronic acid-palmitate nanoparticles to deliver carbonic anhydrase inhibitors can impair Escherichia coli growth and metabolism.

## Contribution

The study demonstrates the effectiveness of HA-PA nanocarriers in delivering CA inhibitors and reveals their impact on bacterial energy homeostasis.

## Key findings

- AZA delivered via HA-PA nanoparticles showed the strongest inhibition of E. coli growth.
- All inhibitors caused an increase in intracellular ATP, suggesting reduced ATP consumption in bicarbonate-dependent pathways.
- HA-PA nanocarriers effectively deliver CA inhibitors intracellularly and enhance their antimicrobial activity.

## Abstract

Bacterial carbonic anhydrases (CAs) are essential for intracellular pH regulation, bicarbonate homeostasis, and energy metabolism, making them attractive antimicrobial targets. Here, building on evidence that acetazolamide (AZA) delivered via hyaluronic acid–palmitate (HA-PA) nanocarriers impairs Escherichia coli growth and its glucose uptake, we investigated the physiological roles of β- and γ-class CAs using sulphonamide inhibitors with distinct selectivity encapsulated in HA-PA nanomicelles to ensure intracellular delivery. AZA, a potent dual β/γ-CA inhibitor, ethoxzolamide (EZA), a selective β-CA inhibitor, and hydrochlorothiazide (HCT), a weaker inhibitor of both classes, were tested for effects on bacterial physiology. The nanoparticles reduced growth in a dose- and class-dependent manner, with AZA exerting the strongest activity, EZA intermediate inhibition, and HCT only modest effects at higher concentrations. Early metabolic responses assessed via intracellular ATP after three hours of exposure revealed an unexpected and reproducible ATP increase for all inhibitors relative to untreated cells, suggesting reduced ATP consumption in bicarbonate-dependent pathways. These findings provide indirect yet compelling evidence that β- and γ-class CAs influence bacterial energy homeostasis and support the rationale for CA inhibition as an antimicrobial strategy, while highlighting HA-PA carriers as effective systems for delivering CA inhibitors intracellularly and enhancing their functional activity in bacterial cells.